FFPE tissue is one of the most important sources of DNA and ribonucleic acid (RNA) for clinical cancer diagnosis and research. Such diagnosis and research is often done using advanced genetic sequencing techniques known as next-generation sequencing (NGS). Diagnostic procedures involving NGS often involve detecting low frequency somatic mutations in a DNA library prepared from one or more FFPE tissue samples, such mutations being key indicators of cancer progression in patients.
One of the more popular methods of preparing such a DNA library involves purifying genomic DNA using the QIAamp® DNA FFPE Tissue Kit distributed by QIAGEN Gmbh. Another popular method involves use of the Ion AmpliSeq® Direct FFPE DNA Kit distributed by Thermo Fisher Scientific Inc. However, all such tissue preparation kits and their accompanying procedures are fraught with challenges.
For example, FIG. 1 shows certain steps of a known method of preparing DNA libraries from FFPE tissue samples. The steps shown in FIG. 1 are similar to certain steps of a library preparation procedure involving the QIAamp® DNA FFPE Tissue Kit. Such procedures often require tissue from multiple slide-mounted FFPE samples. This can quickly exhaust a specimen supply when multiple NGS runs are required. The FFPE slides are also deparaffinized in an aromatic solvent such as xylene. However, the health hazards of xylene are well documented and those with long-term exposure to xylene can develop headaches, dizziness, nausea, and vomiting (see Kandyala, R., Raghavendra, S. P. C., & Rajasekharan, S. T. (2010). Xylene: An overview of its health hazards and preventive measures. Journal of Oral and Maxillofacial Pathology, 14(1):1). Once deparaffinized, the FFPE slides are washed in ethanol to displace the xylene-based solution and the exposed tissue samples on the slides are then scraped off with a scalpel or razor blade and transferred to a test tube or reaction tube. The collected tissue sample is then subjected to a proteinase K digestion step to digest contaminating proteins and a lysis buffer is added to lyse remaining contaminating cellular components. The lysate is then transferred to a reaction tube comprising a mini-column made of silica membrane. Additional buffers are then added to the reaction tube and the entire tube is centrifuged to bind the DNA from the tissue sample to the mini-column. The mini-column is then transferred to a collection tube and an elution buffer is added. The tube is finally centrifuged to elute the DNA from the mini-column. DNA amplification reagents can then be added to the eluted DNA and such a mixture can then be subjected to an amplification reaction to yield the DNA library.
As can be seen from FIG. 1, current methods of preparing DNA libraries from FFPE tissue samples are often laborious and time-consuming (e.g., up to 72 hours). Such methods also require multiple reagents and buffers and are susceptible to high risks of clinician error. While other methods, such as those involving the Ion AmpliSeq® Direct FFPE DNA Kit, have eliminated the deparaffinization step with xylene, such methods require the FFPE tissue sample to be dissolved in mineral oil and require multiple reagents and a heating step to extract DNA from the tissue sample. Additional steps are then required to prepare the extracted DNA for further amplification using reagents from a separate DNA amplification kit.
Therefore, a solution is needed which reduces the number of operational steps needed to prepare DNA libraries from slide-mounted FFPE tissue samples yet maintain or improve the quantity and quality of target sequence yields compared to convention methods. Such a solution should be cost-effective compared to conventional methods, require less time, and should lessen the risk of clinician or operator error.